Genomic Profiling Reveals Differences in Primary Central Nervous System Lymphoma and Large B-Cell Lymphoma, With Subtyping Suggesting Sensitivity to BTK Inhibition

Abstract Background B-cell primary central nervous system (CNS) lymphoma (PCL) is diffuse large B-cell lymphoma (DLBCL) confined to the CNS. Less than 50% of patients with PCL achieve complete remission with current therapies. We describe the findings from comprehensive genomic profiling (CGP) of a cohort of 69 patients with PCL, 36 cases of secondary CNS lymphoma (SCL), and 969 cases of DLBCL to highlight their differences and characterize the PCL cohort. In addition, we highlight the differences in frequency of germinal center B-cell like (GCB) and non-GCB subtypes and molecular subtypes, particularly MCD and EZH subtypes, between PCL and DLBCL. Materials and Methods Sixty-nine cases of B-cell PCL, 36 cases of secondary CNS lymphoma (SCL), and 969 cases of DLBCL were evaluated by CGP of 405 genes via DNAseq and 265 genes via RNAseq for fusions (FoundationOne Heme). Tumor mutational burden (TMB) was calculated from 1.23 Mb of sequenced DNA. Results Genomic alterations with significant differences between PCL and DLBCL included MYD88, ETV6, PIM1, PRDM1, CXCR4, TP53, and CREBBP, while only MYD88 was significantly different between SCL and DLBCL. PCL cases were significantly enriched for the MCD molecular subtypes, which have an excellent response to BTKi. We report a patient with a durable complete response to BTKi consistent with their genomic profile. EBV status, CD274 amplification, and TMB status suggest that 38% of PCL patients may benefit from ICPI; however further study is warranted. Conclusion CGP of PCLs reveals biomarkers, genomic alterations, and molecular classifications predictive of BTKi efficacy and potential ICPI efficacy. Given the limitations of standard of care for PCL, CGP is critical to identify potential therapeutic approaches for patients in this rare form of lymphoma.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most prevalent type of non-Hodgkin lymphoma (NHL), with over 20 000 new cases diagnosed in the US annually. 1 A small subset of DLBCL cases arise within and are restricted to the central nervous system. These are termed primary CNS lymphoma B-cell-like (ABC or non-GCB). The genomic landscape of non-GCB DLBCL has been well characterized, with oncogenic driver mutations typically affecting NF-kB and B-cell receptor (BCR) activation. 5,6 Conversely, GCB DLBCL often involves constitutive activation of BCL2, preventing apoptosis. 5 The differences between the 2 groups have important prognostic implications, as non-GCB DLBCL is a predictor of poor response to R-CHOP. 7 Previous studies have demonstrated that most PCL cases are of the non-GCB subtype. 8 Further subclassification of DLBCL is now possible using genomic profiles using the LymphGEN tool. 9 The most common molecular subtypes are MCD and EZB, which most typically correlate with non-GCB and GCB subtyping respectively. These subtypes are of particular importance as they can predict the outcome of various therapeutic approaches. For example, systemic DLBCL of the MCD subtype have a robust response to BTKi, 16 with 100% 5-year event free survival (PFS) in younger patients when treated with BTKi plus R-CHOP versus <50% with R-CHOP alone).
Sequencing of CNS lymphomas in other cohorts has revealed a consistent spectrum of genomic alterations, specifically highlighting recurrent genomic alterations in the NF-kB pathway (CD79B, MYD88, TNFAIP3, and CARD11), with additional alterations such as loss of CDKN2A, PRDM1, and IgH-BCL6 translocations. [10][11][12][13][14][15][16] Standard-of-care therapy for CNS lymphomas involves chemotherapy (combination methotrexate and R-CHOP). [17][18][19] In general these therapies have limited success, with a median progression-free survival (PFS) of 39 months with a 79% survival rate at 2 years. 20 Historically, CHOP chemotherapy was standard treatment for DLBCL. 21 The addition of rituximab was beneficial to DLBCL outcomes, but those improvements were muted in patients with PCL. 22 Even for patients with PCL who achieve a complete response (CR), most do not have durable responses, with approximately 50% of patients experiencing refractory/relapsed disease. 23,24 The variable outcomes for PCL patients with standard-of-care therapy highlight the need for improved treatment modalities. Promising new therapeutic modalities being explored for PCL clinical management include Bruton tyrosine kinase (BTK) inhibitors (BTKi) and immune checkpoint inhibitors (ICPI)/immunotherapy. Ibrutinib, a BTKi, has performed well in DLBCL clinical trials, particularly in non-GCB patients compared with patients with GCB. 31,32 This preferential effect is a result of the chronic activation of B-cell receptors in non-GCB cells, 6 which ibrutinib targets. Notably, clinical trials of monotherapy ibrutinib in salvage treatment of PCL showed high overall (77%, 10/13) and complete (39%, 5/13) response rates. 33 However, resistance to BTKi monotherapy may be conferred to patients with PCL harboring certain mutations. MYD88, notably MYD88 L265P, has been shown to confer resistance to BTKi in non-GCB DLBCL; combination therapy with histone deacetylation inhibitors has shown some effectiveness in patients with MYD88 genomic alterations. 34 CARD11 mutations in follicular lymphoma confer BTKi resistance. 35 Additionally, investigation into CD79B overexpression revealed induced BTKi resistance through AKT/MAPK pathways in DLBCL. 36 Specifically in patients with the MCD and N1 molecular subtypes of non-GCB DLBCL, BTKi in conjunction with R-CHOP has resulted in significantly improved survival over R-CHOP alone, with a 3-year event free survival of 100% versus <50% for R-CHOP alone. 16 Immunotherapy is a strong candidate across multiple tumor types. Mouse models of immunocompetent PCL have shown increased immune-factor infiltration and improvements in overall survival with ICPI. 25 Moreover, immunohistochemistry of patients with PCL indicate 71% (12/17) of cases score positively for PD-L1, suggesting that ICPI may have value in this patient population. 26 PCL is enriched for 9p24.1 amplification 27 which increases PD-L1 expression; and PCL often has PD-L1 expression (59% of cases (n = 71) in one study 28 ). These both provide an explanation for the increased ICP susceptibility. 29 The use of nivolumab, in one cohort, showed complete responses in 80% (4/5) and 17-month PFS in 60% (3/5) of patients. 30 This study describes the genomic landscape and prevalence of immunotherapy biomarkers, including tumor mutational burden (TMB), in a large cohort of PCL, SCL, and DLBCL patients. GCB and non-GCB status is compared as are the molecular DLBCL subtypes. This is the second largest cohort of PCL cases to date (n = 69), and the largest to compare the molecular subtypes between PCL, SCL, and DLBCL. The purpose is to demonstrate the utility of comprehensive genomic profiling (CGP) for identifying therapies for PCL patients that may be alternatives to standard approaches currently available. The use of CGP for identifying treatment strategies is highlighted by a case study of complete response to ibrutinib in a patient with refractory PCL.

Methods
CGP was performed in a College of American Pathologists (CAP)-accredited, Clinical Laboratory Improvement Amendments (CLIA)-certified, New York State-regulated reference laboratory (Foundation Medicine, Inc.). This study was approved by the Western Institutional Review Board (IRB# 20152817) and includes a waiver of informed consent and a HIPAA waiver of authorization. All samples underwent central histopathologic review by a board-certified pathologist. DNA and RNA were extracted from formalin-fixed paraffin-embedded tumor samples, and next-generation sequencing was performed on hybridization-captured, adaptor ligation-based libraries to high, uniform coverage (>500×) for all coding exons of 406 cancer-related genes using DNAseq, and RNAseq in 265 genes (FoundationOne Heme assay). Sequence data were analyzed for clinically relevant classes of genomic alterations, including base pair substitutions, insertions/deletions, copy number alterations, and rearrangements/fusions. TMB was calculated on up to 1.2 Mb as the number of somatic, coding point mutations and indels per Mb of genome (low: <6; intermediate: [6][7][8][9][10][11][12][13][14][15][16][17][18][19]; high: ≥20 mutations/Mb). Microsatellite instability (MSI) status was determined for each tumor and reported as MSI-Stable, MSI-Ambiguous and MSI-High.
CGP was performed on 69 cases of B-cell PCL, 36 cases of SCL, and 969 cases of DLBCL were evaluated for single nucleotide variants using the FoundationOne Heme assay 37 during routine clinical care. The DLBCL cases were selected based on the disease classification assigned at the time of processing. The DLBCL, PCL, and SCL cases were confirmed by review of the provided supporting documentation (pathology reports) at the time of testing. Genomic alteration frequency comparison was performed using a Fisher's exact test. TMB comparison was performed using an ANOVA Test (P < .05).

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The Oncologist, 2023, Vol. 28,No. 1 GCB and non-CGB classifications were assigned based on Hans algorithm 38 using IHC results from pathology reports received at the time of CGP testing. All PCL and SCL cases were classified if IHC results were available and 100 random systemic DLBCL cases with IHC results were classified.

Durable and Complete Response to BTK Inhibition in Refractory Primary CNS Lymphoma
A 79-year-old female presented with progressive lethargy, confusion, diabetes insipidus, and right sixth nerve palsy. A magnetic resonance imaging (MRI) study with contrast of the brain (Fig. 1A) revealed a right inferior frontal enhancing lesion. A biopsy was performed which revealed a densely cellular lesion comprised of a monotonous population of malignant lymphoid cells, consistent with the diagnosis of PCL (Fig. 1B). IHC studies revealed that the tumor cells expressed CD20, BCL2, and BCL6. Systemic evaluation including bone marrow biopsy was negative and CGP was performed on the diagnostic brain biopsy.
CGP of PCL tissues revealed the tumor to be wildtype for CD79B, MYD88, CARD11, and TNFAIP3, with genomic alterations detected in SMO, CIITA, ETS1, HST1H1D, and Table S1). The tumor was found to be hypermutated with a tumor mutational burden of 28 mutations/Mb and was microsatellite stable.
The patient was treated with rituximab, methotrexate (3 g/m 2 ), and temozolomide (100 mg/m 2 ) on a 5/28 schedule with twice weekly intraommaya rituximab (25 mg) but progressed after the seventh cycle (4 months) (Fig. 1C). As a secondary line of treatment, ibrutinib (560 mg daily) and rituximab were initiated, with complete response noted after 5 months (Fig. 1D). She completed 12 months of ibrutinib, with rituximab discontinued due to cytopenias. Since completion of ibrutinib treatment, she has been followed for >5 years with observation only and remains clinically stable, asymptomatic and without evidence of recurrent enhancement on neuroimaging studies (Fig. 1E). A complete response to ibrutinib is consistent with prior reports for patients lacking pathogenic alterations in CD79B, MYD88, CARD11, and TNFAIP3. 33

Classification of PCL, SCL, and DLBCL Cases With Hans Algorithm
As determined by Hans algorithm, 38 significantly more PCL cases were of the non-GCB subtype compared with both the SCL (P < .02) and systemic DLBCL cases (P < .005). SCL and systemic DLBCL cases are not significantly different (P = .89). Seventy-two percent (36/50) of PCL samples were of the non-GCB subtype, 44% (11/25) of SCL samples were of the non-GCB subtype, and 47% (47/100) of systemic DLBCL samples evaluated were of the non-GCB subtype (Fig. 3C).   (Fig. 4D). No other comparisons between molecular subtypes were statically significant.
Of the cases that had IHC results to perform a Hans classification, 30% (15 of 50) PCL cases, 44% (11 of 25) SCL cases, and 43% (43 of 100). Systemic DLBCL cases also were able to be classified by LymphGEN. Across all disease types, 59.6% (28 of 94) of non-GCB and 51.9% (42 of 81) of GCB cases had a classification assigned by LymphGEN. This difference is likely for 2 reasons. First, many MCD cases, which are predominantly non-GCB, were just below the confidence level for classification. If the confidence for calling MCD is lowered from a 0.5 probability to a 0.3 probability, an additional 32 cases are classified as MCD, which raises the percent of non-GCB cases with a classification to 36% (34 of 94). In addition, most A53 cases are of the non-GCB subtype, which are rarely called in the absence of chromosomal arm level data.

Discussion
CGP has the potential to suggest therapeutic strategies of value to patients, or to flag those likely to be ineffective. Given the variable outcomes for PCL patients, the ability to personalize treatment based on defined biomarkers is critical. In this study, we examined the genomic landscape of PCL, molecular subtyping of PCL, and the distribution of biomarkers such as TMB and explored how those results might be targeted by emerging therapeutic strategies. Our survey of this patient population found that PCL cases were significantly more likely than SCL or systemic DLBCL samples to be of the non-GCB subtype (72% vs 44% and 47%, respectively)), as determined by reported IHC results using Hans algorithm, which is consistent with other studies characterizing PCLs 41 and genomic alterations.
Molecular classification of DLBCL cases, whether PCL, SCL, or systemic DLBCL is valuable and avoids some of the pitfalls of the Hans classification. One difficulty with Hans classification, especially at a reference laboratory is the heterogeneity of IHC results as reported across different institutions. Data available from CGP only was sufficient to classify approximately 50% of cases. This is compared to 63% of cases that were able to be identified in the Wright et al study. 9 Integration of arm level chromosomal gains and losses from karyotyping and additional FISH results unavailable in a reference lab setting likely account for the difference and could be integrated in clinical practice. In particular, the A53 subtype was rarely called (2 of 1058 cases) without chromosomal arm level data, compared to the expected rate of 6% in the presence of chromosomal arm level data, which accounts for at least half of that difference.

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Of interest, while SCL and systemic DLBCL had similar classification results based on Hans classification, both PCL and SCL were significantly more likely to be of the MCD molecular subtype than systemic DLBCL, while systemic DLBCL was significantly more likely to be of the EZB subtype. Of note, all PCL cases that had both Hans classification were of the MCD subtype, while some of those cases were determined to be GCB subtype with Hans classification, which highlights the difficulties of using IHC results abstracted from pathology reports across many different institutions. Per Wright et al nearly all cases classified as MCD should be of the non-GCB subtype.
Of the cases that had IHC results to perform a Hans classification, 30% of non-GCB cases could be classified compared to 52% of GCB cases. This difference is likely for 2 reasons. First, many MCD cases, which are predominantly non-GCB, were just below the confidence level for classification. If the confidence for calling MCD is lowered from a 0.5 probability to a 0.3 probability, an additional 32 cases are classified as MCD, which raises the percent of non-GCB cases with a classification to 36% (34 of 94). In addition, most A53 cases are of the non-GCB subtype, which are rarely called in the absence of chromosomal arm level data.
Molecular subtyping of this kind has significant prognostic value, both for predicting overall patient outcomes but also for determining the potential responsiveness to different therapies. BKTi represents one such directed therapy, which is more efficacious in non-GCB DLBCLs such as PCL. This was specifically demonstrated for systemic DLBCL in Wright et al where excellent response of the MCD and N1 subgroups to BTKi was demonstrated. 9 Systemic DLBCL of the subtypes MCD and N1 have a robust response to BTKi, 16 with 100% 5-year event free survival (PFS) in younger patients when treated with BTKi plus R-CHOP versus <50% with R-CHOP alone). This is of particular importance in PCL, where there the MCD subtype is significantly enriched.
There are resistance mechanisms that allow tumors to bypass BTK inhibition. In a phase I clinical trial, BTK inhibition with ibrutinib resulted in a complete response in patients with wild-type CD79B, CARD11, and TNFAIP3, with partial or complete resistance associated with GAs in any of these B-cell receptor pathway genes. 33 MYD88 L265P has been characterized in PCL as being associated with resistance to BTKi monotherapy. 34,42 Overexpression of CD79B can induce BTKi resistance by enhancing AKT/MAPK function in DLBCL. 32,36 One clinical trial in PCL using BTKi in PCL also implicated CARD11 and TNFAIP3, being members of the NF-kB pathway, as potential means of resistance development. 33 The results of clinical trials based on molecular subtypes for BTKi in systemic DLBCL merits caution in interpreting these earlier findings and indicates that specific studies of PCL and BTKi based on molecular subtyping should be performed.
ICPIs, such as nivolumab or pembrolizumab, have also shown value as novel therapeutic strategies, particularly against oncogenic targets with immune-evading mutations. In one Hodgkin's lymphoma cohort, patients showed an 87% (20/23) response rate to nivolumab. 43 Similarly, patients with 9p24.1 amplifications with relapsed PCL all (5/5) showed clinical and radiographic responses, with most (3/5) showing diseasefree progression after 17 months. 44 In addition to general effectiveness, several genomic biomarkers are associated with the effectiveness of ICPIs. Clinical studies have suggested that TMB may be useful in predicting the outcome of immunotherapy when looking at non-small cell lung cancer 45 ; however, not all tumor types benefit from immunotherapy with high TMB. 15 Additionally, EBV+ cells of lymphoproliferative disorders have been shown to express PD-L1 in immunocompetent patients, which may indicate immunotherapy susceptibility. 46,47 CGP of PCLs in this study revealed many samples harbored biomarkers indicative of potential immunotherapeutic susceptibility, including high TMB (24/69, 34.8%), CD274 amplification (4/69, 5.8%), Chr9 or Chr9p gains (11/69, 10.1%), and/or EBV positivity (6/69, 8.7%), affecting 49.3% (34/69) of patients in total. These data indicate that these patients could potentially show enhanced responses to ICPIs. Further work should be done specifically in PCL to investigate the efficacy of immunotherapy in relation to these genomic biomarkers.
Primary CNS lymphoma (PCL) is a genomically distinct type of DLBCL and has a poor prognosis with standard therapies. For newer treatments, comprehensive genomic profiling (CGP) and molecular subtype assignment is critical, as it reveals biomarkers and classifications that may be predictive of immunotherapy efficacy (TMB-high, CD274 amplification) and molecular classifications, specifically MCD and N1, that are predictive of BTKi efficacy. Given the limitations of standard of care for PCL, CGP can direct patients to newer therapeutic approaches in this rare form of lymphoma.

Funding
None declared.